Method for detecting cell proliferative disorders

ABSTRACT

The present invention relates to the detection of a cell proliferative disorder associated with alterations of microsatellite DNA in a sample. The microsatellite DNA can be contained within any of a variety of samples, such as urine, sputum, bile, stool, cervical tissue, saliva, tears, or cerebral spinal fluid. The invention is a method to detect an allelic imbalance by assaying microsatellite DNA. Allelic imbalance is detected by observing an abnormality in an allele, such as an increase or decrease in microsatellite DNA which is at or corresponds to an allele. An increase can be detected as the appearance of a new allele. In practicing the invention, DNA amplification methods, particularly polymerase chain reactions, are useful for amplifying the DNA. DNA analysis methods can be used to detect such a decrease or increase. The invention is also a method to detect genetic instability of microsatellite DNA. Genetic instability is detected by observing an amplification or deletion of the small, tandem repeat DNA sequences in the microsatellite DNA which is at or corresponds to an allele. The invention is also a kit for practicing these methods.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present invention claims priority under 35 U.S.C. §119(e)from Provisional Application Serial Number 60/025,805, filed Aug. 28,1996.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the detection of atarget nucleic acid sequence and specifically to the detection ofmicrosatellite DNA sequence mutations associated with a cellproliferative disorder.

[0004] 2. Description of Related Art

[0005] Cancer remains a major cause of mortality worldwide. Despiteadvancements in diagnosis and treatment, the overall survival rate hasnot improved significantly in the past twenty years. There remains anunfulfilled need for a more sensitive means of early diagnosis oftumors, before the cancer progresses.

[0006] One of the most serious cancers is bladder cancer. Bladder canceris the fourth most common cancer in men and the eighth most common inwomen. Transitional cell carcinoma (TCC) of the bladder is the mostcommon urothelial malignancy of the urinary tract, with an incidence ofapproximately 51,000 each year in the United States alone.

[0007] One reason that bladder cancer is so serious is because,presently, detecting and treating bladder cancer is difficult. Seventypercent of patients with an initial diagnosis of transitional cellcarcinoma have superficial tumors, which can be treated by transurethralresection alone. Approximately 70% of these patients continue to sufferfrom recurrent disease, and 15% develop lesions that invade musclewithin the first two years.

[0008] Detecting tumor recurrence in patients with transitional cellcarcinoma of the bladder requires close surveillance. Urine cytology isa common non-invasive procedure for the diagnosis of this disease, butit can miss up to 50% of tumors. The “gold standard” for diagnosis iscystoscopy, which allows visualization and direct biopsies of suspiciousbladder lesions in the mucosa. However, because cystoscopy is anexpensive and invasive procedure, it cannot be used as a generalscreening tool for the detection of bladder cancer.

[0009] Other serious cancers are the head and neck cancers. Head andneck cancer remains a morbid and often fatal disease. Large tumor bulkand tumor extension are predictors of a local regional recurrence andpoor outcome. Detection of occult neoplastic cells in surroundingsurgical margins is a strong predictor of local regional recurrenceresulting in a significant decrease in overall survival.

[0010] DNA contains unique sequences interspersed with moderately andhighly repetitive DNA sequences. Variations in the repetitive sequenceelements such as minisatellite (or variable number tandem repeat) DNAsequences and microsatellite (or variable simple sequence repeat) DNAsequences have been useful for chromosomal identification, primary genemapping, and linkage analysis. Microsatellite DNA sequences are anespecially common and highly polymorphic class of genomic elements inthe human genome. One advantage to the use of repetitive sequencevariations is the greater number of alleles present in populationscompared with unique genetic sequence variations. Another advantage isthe ability to readily detect sequence length variations using thepolymerase chain reaction for the rapid and inexpensive analysis of manyDNA samples.

[0011] Tumors progress through a series of genetic mutations. Thesegenetic mutations can be used as specific markers for the detection ofcancer. One set of genetic mutations that can be used to detect thepresence of cancer is the loss of chromosomes. Diploid organisms,including humans, have pair of chromosomes for each member of thechromosomal set. Tumor cells will characteristically lose chromosomes,resulting in a single chromosome, rather than a pair of chromosomes, foreach member of the chromosomal set. Chromosomal deletions and additionsare an integral part of neoplastic progression and have been describedin most kinds of cancers. A pair of chromosomes has two alleles for agenetic locus is heterozygous for that locus; therefore, theheterozygosity correlates to the cell having a pair of chromosomes. Foryears, these chromosomal deletions or amplifications were detectedthrough the loss of heterozygosity.

[0012] Another of the genetic mutations used to detect the presence ofcancer is genetic instability. Genetic recombination tends to occur mostfrequently at regions of the chromosome where the DNA is homologous(where the DNA has a high degree of sequence similarity). Where a DNAsequence is repetitive, the DNA homology is greater. The DNA homologyoccurs not only at the same genetic locus on the other pair ofchromosomes, but also on other genetic loci or within the same locus onthe same chromosome. Normal (non-tumor) cells tend to suppress thisgenetic recombination. Tumor cells, however, characteristically undergoincreased genetic recombination. Where a DNA sequence is repetitive,genetic recombination can result in the loss of repeat DNA sequences orthe gain of repeat DNA sequences at a genetic locus.

[0013] Microsatellite DNA instability has been described in humancancers. Microsatellite DNA instability is an important feature oftumors from hereditary non-polyposis colorectal carcinoma patients(Peltomaki et al., Science, 260: 810 (1993); Aaltonen et al., Science,260: 812 (1993); Thibodeau et al., Science, 260: 816 (1993)).Microsatellite DNA instability by expansion or deletion of repeatelements has also been reported in colorectal, endometrial, breast,gastric, pancreatic, and bladder neoplastic tissues (Risinger et al.,Cancer Res., 53: 5100 (1993); Had et al., Cancer Res., 53: 5087 (1993);Peltomaki et al., Cancer Res. 53: 5853 (1993); Gonzalez-Zulueta et al.,Cancer Res. 53: 5620 (1993)).

[0014] Some methods have been developed to detect the multiple geneticchanges that occur during the development of primary bladder cancer. Forexample, mutations in the tumor suppressor gene p53 signal theprogression to invasiveness and have been successfully used as molecularmarkers to detect cancer cells in urine samples. However, thisdiagnostic strategy has limited clinical application because thetechniques are cumbersome and because p53 mutations appear relativelylate in the disease.

[0015] Because early diagnosis of bladder cancer is critical forsuccessful treatment, there is a pressing need for more sensitive andcost-effective diagnostic tools. Both patients and physicians wouldbenefit from the development of improved non-invasive methods for cancersurveillance.

SUMMARY OF THE INVENTION

[0016] The present invention provides a fast, reliable, sensitive andnon-invasive screening method for the detection of a cell proliferativedisorder in a subject. The method detects an allelic imbalance byassaying microsatellite DNA, wherein an abnormality in an allele isindicative of an allelic imbalance. Such abnormalities include anincrease or decrease in microsatellite DNA that is at or corresponds toan allele. A decrease can be detected such that the level of DNAcorresponding to the allele is less than 50% of the level of DNA of acorresponding allele in a microsatellite DNA sample of a subject thatlacks the cell proliferative disorder. An increase can be detected asthe appearance of a new allele.

[0017] The cell proliferative disorder detected by the method of theinvention may be a neoplasm, for example, a neoplasm of the head, neck,lung, esophageal, stomach, small bowel, colon, bladder, kidney, orcervical tissue. The sample of microsatellite DNA may be urine, sputum,bile, stool, cervical tissue, saliva, tears, cerebral spinal fluid,serum, plasma, or lymphocytes, for example.

[0018] The microsatellite DNA detected by the method may be a locus suchas DRPLA, UT762, IFNA, D9S200, D9S156, D3S1284, D3S1238, CHRNB1, D17S86,D9S747, D9S171, D16S476, D4S243, D14S50, D21S1245, FgA, D8S3G7, THO,D115488, D135802, D175695, D175654, and D20548.

[0019] The invention also provides a fast, reliable, sensitive andnon-invasive screening method for detecting genetic instability ofmicrosatellite DNA. An amplification or deletion of the small tandemrepeat DNA sequences indicates genetic instability in the microsatelliteDNA that is at or corresponds to an allele.

[0020] The present invention also provides a kit for detecting a cellproliferative disorder, comprising oligonucleotide primers that arecomplementary to a nucleotide sequence that flanks nucleotide repeats ofmicrosatellite DNA. In one embodiment, the kit further comprises adetectably labeled deoxyribonucleotide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 is a table of patients with bladder lesions and urineanalysis (Cyto, cytology; LOH, loss of heterozygosity; Alt,microsatellite).

[0022]FIG. 2 is a table of microsatellite analysis of urine sediment.

[0023]FIG. 3 is a table of microsatellite analysis and clinical outcomein head and neck cancer patients.

[0024]FIG. 4 is a table of microsatellite analysis of patients withalterations in plasma.

[0025]FIG. 5 is a table of cytology and molecular status of patientswith bladder cancers.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention relates to a non-invasive method for thedetection of a cell proliferative disorder associated with mutations ofmicrosatellite DNA in a subject. The invention provides a method fordetecting an allelic imbalance by assaying microsatellite DNA. Theinvention also provides a method for detecting genetic instability ofmicrosatellite DNA. The present invention is based on the threefollowing principles:

[0027] First, chromosomal deletions and genetic recombination are anintegral part of neoplastic progression and have been described in mostkinds of cancers. Allelic imbalance (loss of heterozygosity) and geneticinstability can now be detected in clinical samples composed mostly ofnormal-looking (morphologically normal) cells. The clinical samples canbe readily obtained, thus providing a non-invasive alternative tosurgery and microdissection of neoplastic tissue. Second, monoclonalityis a fundamental characteristic of neoplasms. Clonal genetic mutationsare integrally involved in the progression of all cancers. Detection ofa clonal population of cells harboring a chromosomal deletion oramplification is synonymous with the detection of cancer at a molecularlevel.

[0028] Third, microsatellite DNA in clinical samples can be amplified invitro to detect an allelic imbalance (loss of heterozygosity) or geneticinstability of microsatellite DNA. A combination of markers for eachtumor type can now be used to identify many tumors in a given clinicalsample. Moreover, these markers can also be multiplexed in a singleamplification reaction to generate a low cost, reliable cancer screeningtest for many cancers simultaneously.

[0029] In one embodiment, the invention provides a method for detectinga cell proliferative disorder in a subject by detecting, in a sample ofmicrosatellite DNA from the subject, an allelic imbalance. The presenceof an allelic imbalance is indicative of a cell proliferative disorder.

[0030] The term “cell-proliferative disorder” includes both benign andmalignant cell populations that morphologically differ from thesurrounding tissue. For example, the method is useful for detectingtumors of the lung, breast, lymphoid, gastrointestinal, andgenitourinary tract; epithelial carcinomas that include malignanciessuch as most colon cancers, renal-cell carcinoma, prostate cancer,non-small cell carcinoma of the lung, cancer of the small intestine,stomach cancer, kidney cancer, cervical cancer, cancer of the esophagus,and any other organ type that has a draining fluid or tissue accessibleto analysis; and nonmalignant cell-proliferative diseases such as colonadenomas, hyperplasia, dysplasia and other pre-malignant lesions. Anydisorder that is etiologically linked to mutations in a microsatelliteDNA locus is susceptible to detection. In one embodiment, the method ofthe invention is useful for the detection of transitional cell carcinomaof the bladder and for the detection of head and neck cancer.

[0031] A cell proliferative disorder as described herein may be aneoplasm. Such neoplasms are either benign or malignant. The term“neoplasm” refers to a new, abnormal growth of cells or a growth ofabnormal cells that reproduce faster than normal. A neoplasm creates anunstructured mass (a tumor) which can be either benign or malignant. Forexample, the neoplasm may be a head, neck, lung, esophageal, stomach,small bowel, colon, bladder, kidney, or cervical neoplasm. The term“benign” refers to a tumor that is noncancerous, e.g. its cells do notproliferate or invade surrounding tissues. The term “malignant” refersto a tumor that is metastastic or no longer under normal cellular growthcontrol.

[0032] The term “allelic imbalance” refers to the chromosomal loss orgain that is characteristic of tumor cells. Diploid organisms, includinghumans, have a pair of chromosomes for each member of the chromosomalset. Tumor cells characteristically lose chromosomes, often resulting ina single chromosome, rather than a pair of chromosomes, for each memberof a chromosomal set. Tumor cells also on occasion gain chromosomes,resulting in a two or more chromosomes, rather than a pair ofchromosomes, for each member of the chromosomal set.

[0033] When a genetic locus on the chromosome has a different DNAsequence on each chromosome, a diploid organism has two alleles for thatgenetic locus. A pair of chromosomes with two alleles for a geneticlocus is heterozygous. Whether a genetic locus is heterozygous for asubject can readily be determined by analyzing a sample of DNA from thenormal (non-tumor) cells of the subject. Because microsatellite DNA ispolymorphic, a genetic locus that contains microsatellite DNA willfrequently be heterozygous. When a tumor cell loses or gains achromosome, the result is that the cell loses or gains an additionalcopy of one of the alleles, causing an allelic imbalance (loss ofheterozygosity).

[0034] Microsatellite DNA markers that are heterozygous in normal(non-tumor) cell DNA can be used to detect mutations in tumor cell DNA.The loss of one allele identifies chromosomal deletions after gelelectrophoresis or other techniques. An imbalance between the twoalleles also identifies chromosomal amplifications. To do these analysesby conventional methods requires extensive microdissection of neoplasticcells so that normal (non-tumor) contaminating cells would not disruptthe assay. The method of the invention, by contrast, provides anon-invasive sampling technique in which the presence of normal(non-tumor) cells does not interfere with the assay. A loss ofheterozygosity correlating with bladder cancer can be detected in urinesamples in patients where the samples contain both normal and tumorcells. A loss of heterozygosity can be detected in the plasma and salivaof patients with head and neck cancer.

[0035] A combination of microsatellite DNA markers may be amplified in asingle amplification reaction. The markers are multiplexed in a singleamplification reaction, for example, by combining primers for more thanone locus. For example, DNA from a urine sample can be amplified withthree different randomly labeled primer sets, such as those used for theamplification of the FgA, ACTBP2 and AR loci, in the same amplificationreaction. The reaction products are separated on a denaturingpolyacrylamide gel, for example, and then exposed to film forvisualization and analysis.

[0036] The term “microsatellite DNA” refers to mononucleotide,dinucleotide, or trinucleotide sequences where alleles differ by one ormore repeat units. Microsatellite DNA is an especially common and highlypolymorphic class of genomic elements in the human genome. Themicrosatellite DNA most preferred in the method of the invention has asequence (X)_(n), wherein X is the number of nucleotides in the repeatsequence and is greater than or equal to 1, preferably greater than orequal to 2, and most preferably greater than or equal to 3 and wherein nis the number of repeats and is greater than or equal to 2, andpreferably from 4 to 6. When X is 2, the nucleotide sequence may be TC.When X is 3, the nucleotide sequence may be selected from AGC, TCC, CAG,CAA, and CTG. Two examples of trinucleotide repeats are D1S50 and DRPLAmarkers. Preferably when X is 4, the nucleotide sequence may be selectedfrom AAAG, AGAT and TCTT. Two examples of tetranucleotide repeats areincluded in D21S1245 and FgA markers.

[0037] The microsatellite DNA sequence may be genetically linked to aunique locus. For example, microsatellite DNA mutations may be detectedusing a marker selected from ARA (chromosome X), D14S50 (chromosome 14),AR (chromosome X), MD (chromosome 19), SAT (chromosome 6), DRPLA(chromosome 12), ACTBP2 (chromosome 6), FgA (chromosome 4), D4S243(chromosome 4), and UT762 (chromosome 21). Tandem repeat sequences havebeen identified as associated with Huntington's disease (HD), fragile Xsyndrome (FX), myotonic dystrophy (MD), spinocerebellar ataxia type I(SCA1), spino-bulbar muscular dystrophy, and hereditarydentatorubralpallidoluysian atrophy (DRPLA).

[0038] The term “sample of microsatellite DNA” refers to DNA present inor prepared from any tissue of a subject. The nucleic acid from anyspecimen, in purified or nonpurified form, can be used as the startingnucleic acid or acids, provided it contains, or is suspected ofcontaining, the specific nucleic acid sequence containing the targetnucleic acid. Thus, the process may employ, for example, DNA or RNA,including messenger RNA (mRNA). The DNA or RNA may be single stranded ordouble stranded. When RNA is used as a template, enzymes and conditionsoptimal for reverse transcribing the template to DNA would be used. ADNA-RNA hybrid that contains one strand of each may also be used. Amixture of nucleic acids may also be employed, or the nucleic acidsproduced in a previous amplification reaction herein, using the same ordifferent primers may be so used. The mutant nucleotide sequence to beamplified may be a fraction of a larger molecule or can be presentinitially as a discrete molecule, such that the specific sequence is theentire nucleic acid. It is not necessary that the sequence to beamplified be present initially in a pure form; it may be a minorfraction of a complex mixture, such as contained in whole human DNA.

[0039] Samples or specimens include any microsatellite DNA sequence,whatever the origin, as long as the sequence is detectably present in asample. While routine diagnostic tests may not be able to identifycancer cells in these samples, the non-invasive method of the presentinvention identifies neoplastic cells derived from the primary tumor.The sample of microsatellite DNA of the subject may be serum, plasma,lymphocytes, urine, sputum, bile, stool, cervical tissue, saliva, tears,cerebral spinal fluid, regional lymph node, histopathologic margins, andany bodily fluid that drains a body cavity or organ. Therefore, themethod provides for the non-invasive detection of various tumor typesincluding head and neck cancer, lung cancer, esophageal cancer, stomachcancer, small bowel cancer, colon cancer, bladder cancer, kidneycancers, cervical cancer and any other organ type that has a drainingfluid accessible to analysis. For example, neoplasia of regional lymphnodes associated with a primary mammary tumor can be detected using themethod of the invention. Regional lymph nodes for head and neckcarcinomas include cervical lymph nodes, prelaryngeal lymph nodes,pulmonary juxtaesophageal lymph nodes and submandibular lymph nodes.Regional lymph nodes for mammary tissue carcinomas include the axillaryand intercostal nodes. Samples also include urine DNA for bladder canceror plasma or saliva DNA for head and neck cancer patients.

[0040] The method of the invention can also be used to detect amicrosatellite DNA sequences associated with a primary tumor by assayingthe surrounding tumor margin. A “tumor margin” as used herein refers tothe tissue surrounding a discernible tumor. In the case of surgicalremoval of a solid tumor, the tumor margin is the tissue cut away withthe discernible tumor that appears normal to the naked eye.

[0041] An allelic imbalance may be detected as a decrease in the levelof DNA corresponding to an allele. The term “decrease in the level ofDNA” refers to the observed difference of the ratio between the twoalleles for a genetic locus. A sample of cells can have a ratioapproaching 1:1 for a subject that lacks the cell proliferativedisorder. The actual ratio for a subject can readily be determined byanalyzing the DNA from the normal (non-tumor) cells of the testedsubject. The level of DNA corresponding to the allele may be less than50% of the level of DNA of a corresponding allele in a microsatelliteDNA sample of a subject that lacks the cell proliferative disorder.

[0042] An allelic imbalance may also be detected as an increase in thelevel of DNA corresponding to an allele. The term “increase in the levelof DNA” refers to the observed difference of the ratio between the twoalleles for a genetic locus. A sample of cells will have a ratio thatapproaches 1:1 for a subject that lacks the cell proliferative disorder.Analyzing the DNA from the normal (non-tumor) cells of a test subjectcan readily determine the actual ratio for the subject.

[0043] An increase in the level of DNA may be detected as the appearanceof a new allele. The term “presence of a new allele” refers both refersto the observed difference of the ratio between the two alleles for agenetic locus and to genetic instability, the genetic recombinationsthat are characteristic of tumor cells and that result in nucleic acidmutations as described infra. When there has been an increase in thenumber of chromosomes for a member of the chromosomal set, one of thechromosomes may undergo genetic recombination, so that there will be anaddition or deletion of DNA repeats in the microsatellite DNA sequence.The mutated microsatellite DNA sequence is therefore a new allele forthat genetic locus, as compared with the normal (non-tumor) cells of thesubject. For example, a tumor cell may have three or more differentalleles for a genetic locus instead of the two alleles found in thenormal (non-tumor) cells. For another example, the presence of a newallele may correspond to the loss of an allele found in normal(non-tumor) cells.

[0044] Detection of an allelic imbalance may be performed by standardmethods such as size fractionating the DNA. The term “size fractionatingthe DNA” refers to the separation of individual DNA molecules accordingto the size of the molecule. Methods of fractionating the DNA are wellknown to those of skill in the art. Fractionating the DNA on the basisof size may be accomplished by gel electrophoresis, includingpolyacrylamide gel electrophoresis (PAGE). For example, the gel may be adenaturing 7 M or 8 M urea-polyacrylamide-formamide gel. Sizefractionating the DNA may also be accomplished by chromatographicmethods known to those of skill in the art.

[0045] The reaction products containing microsatellite DNA mayoptionally be radioactively labeled. Any radioactive label may beemployed which provides an adequate signal. Other labels includeligands, which can serve as a specific binding pair member for a labeledligand, and the like. The labeled preparations are used to probe nucleicacid by the Southern hybridization technique, for example. Testnucleotide fragments are transferred to filters that bind nucleic acid.After exposure to the labeled microsatellite DNA probe, which willhybridize to nucleotide fragments containing target nucleic acidsequences, the binding of the radioactive probe to target nucleic acidfragments is identified by autoradiography (see Genetic Engineering, 1,ed. Robert Williamson, Academic Press (1981), pp. 72-81). The particularhybridization technique is not essential to the invention. Severalhybridization techniques are well known or easily ascertained by one ofordinary skill in the art. As improvements are made in hybridizationtechniques, they can readily be applied in the method of the invention.This technique provides a further method of identification that can beadditional or an alternative to size fractionation.

[0046] The microsatellite DNA may be amplified before detecting. Theterm “amplified” refers to the process of making multiple copies of DNAfrom a single molecule of DNA by genetic duplication. The amplificationof DNA may occur in vivo by cellular mechanisms. The amplification ofDNA may also occur in vitro by biochemical processes known to those ofskill in the art. The amplification agent may be any compound or systemthat will function to accomplish the synthesis of primer extensionproducts, including enzymes. Suitable enzymes for this purpose include,for example, E. coli DNA polymerase I, Taq polymerase, Klenow fragmentof E. coli DNA polymerase I, T4 DNA polymerase, other available DNApolymerases, polymerase muteins, reverse transcriptase, ligase, andother enzymes, including heat-stable enzymes (i.e., those enzymes thatperform primer extension after being subjected to temperaturessufficiently elevated to cause denaturation). Suitable enzymes willfacilitate combination of the nucleotides in the proper manner to formthe primer extension products that are complementary to each mutantnucleotide strand. Generally, the synthesis will be initiated at the 3′end of each primer and proceed in the 5′ direction along the templatestrand, until synthesis terminates, producing molecules of differentlengths. There may be amplification agents, however, that initiatesynthesis at the 5′ end and proceed in the other direction, using thesame process as described above. In any event, the method of theinvention is not to be limited to the embodiments of amplificationdescribed herein.

[0047] One method of in vitro amplification which can be used accordingto this invention is the polymerase chain reaction (PCR) described inU.S. Pat. Nos. 4,683,202 and 4,683,195. The term “polymerase chainreaction” refers to a method for amplifying a DNA base sequence using aheat-stable DNA polymerase and two oligonucleotide primers, onecomplementary to the (+)-strand at one end of the sequence to beamplified and the other complementary to the (−)-strand at the otherend. Because the newly synthesized DNA strands can subsequently serve asadditional templates for the same primer sequences, successive rounds ofprimer annealing, strand elongation, and dissociation produce rapid andhighly specific amplification of the desired sequence. The polymerasechain reaction is used to detect the existence of the defined sequencein the microsatellite DNA sample. Many polymerase chain methods areknown to those of skill in the art and may be used in the method of theinvention. For example, DNA can be subjected to 30 to 35 cycles ofamplification in a thermocycler as follows: 95° C. for 30 sec, 52° to60° C. for 1 min, and 72° C. for 1 min, with a final extension step of72° C. for 5 min. For another example, DNA can be subjected to 35polymerase chain reaction cycles in a thermocycler at a denaturingtemperature of 95° C. for 30 sec, followed by varying annealingtemperatures ranging from 54-58° C. for 1 min, an extension step at 70°C. for 1 min and a final extension step at 70° C.

[0048] Exemplary target nucleotide sequences of the invention, to whichcomplementary oligonucleotide primers hybridize, include the following:5′-CTTGTGTCCCGGCGTCTG-3′ SEQ ID NO:1 5′-CAGCCCAGCAGGACCAGTA-3′ SEQ IDNO:2 5′-TGGTAACAGTGGAATACTGAC-3′ SEQ ID NO:3 5′-ACTGATGCAAAAATCCTCAAC-3′SEQ ID NO:4 5′-GATGGGCAAACTGCAGGCCTGGGAAG-3′ SEQ ID NO:55′-GCTACAAGGACCCTTCGAGCCCCGTT-3′ SEQ ID NO:65′-GATGGTGATGTGTTGAGACTGGTG-3′ SEQ ID NO:75′-GAGCATTTCCCCACCCACTGGAGG-3′ SEQ ID NO:8 5′-GTTCTGGATCACTTCGCGGA-3′SEQ ID NO:9 5′-TGAGGATGGTTCTCCCCAAG-3′ SEQ ID NO:105′-AGTGGTGAATTAGGGGTGTT-3′ SEQ ID NO:11 5′-CTGCCATCTTGTGGAATCAT-3′ SEQID NO:12 5′-CTGTGAGTTCAAAACCTATGG-3′ SEQ ID NO:135′-GTGTCAGAGGATCTGAGAAG-3′ SEQ ID NO:14 5′-GCACGCTCTGGAACAGATTCTGGA-3′SEQ ID NO:15 5′-ATGAGGAACAGCAACCTTCACAGC-3′ SEQ ID NO:165′-TCACTCTTGTCGCCCAGATT-3′ SEQ ID NO:17 5′-TATAGCGGTAGGGGAGATGT-3′ SEQID NO:18 5′-TGCAAGGAGAAAGAGAGACTGA-3′ SEQ ID NO:195′-AACAGGACCACAGGCTCCTA-3′ SEQ ID NO:20 5′-TCTCTTTCTTTCCTTGACAGGGTC-3′SEQ ID NO:21 5′-CAGTGTGGTCCCAAATTTGAAATGG-3′ SEQ ID NO:225′-GTGCTGACTAGGGCAGCTT-3′ SEQ ID NO:23 5′-TGTGACCTGCACTCGGAAGC-3′ SEQ IDNO:24 5′-CCTTTCCTTCCTTCCTTCC-3′ SEQ ID NO:255′-CACAGTCAGGTCAGGCTATCAG-3′ SEQ ID NO:26 5′-TTTTTGAGATAGAGTCTCACTGTG-3′SEQ ID NO:27 5′-CCACAGTCTAAGCCAGTCTGA-3′ SEQ ID NO:285′-GAATTTTGCTCTTGTTGCCCAG-3′ SEQ ID NO:29 5′-AGACTGAAGTCAATGAACAACAAC-3′SEQ ID NO:30 5′-GGCTGTGAACATGGCCTAGGTC-3′ SEQ ID NO:315′-TTGGGGTGGTGCCAATGGATGTC -3′ SEQ ID NO:32

[0049] Exemplary oligonucleotide primers, of the invention, include thefollowing: SEQ ID NO:33 5′-CAGACGCCGGGACACAAG-3′ SEQ ID NO:345′-TACTGGTCCTGCTGGGCTG-3′ SEQ ID NO:35 D21S1245(F)5′-GTCAGTATTACCCTGTTACCA-3′ SEQ ID NO:36 D21S1245(R)5′-GTTGAGGATTTTTGCATCAGT-3′ SEQ ID NO:375′-.CTTCCCAGGCCTGCAGTTTGCCCATC-3′ SEQ iD NO:385′-GAACGGGGCTCGAAGGGTCCTTGTAGC-3′ SEQ ID NO:39 DRPLA(F)5′-CACCAGTCTCAACACATCACCATC-3′ SEQ ID NO:40 DRPLA(R)5′-CCTCCAGTGGGTGGGGAAATGCTC-3′ SEQ ID NO:41 5′-TCCGCGAAGTGATCCAGAAC-3′SEQ ID NO:42 5′-CTTGGGGAGAACCATCCTCA-3′ SEQ ID NO:43 D14S50(F)5′-AACACCCCTAATTCACCACT-3′ SEQ ID NO:44 D14S50(R)5′-ATGATTCCACAAGATGGCAG-3′ SEQ ID NO:45 FgA(F)5′-CCATAGGTTTTGAACTCACAG-3′ SEQ ID NO:46 FgA(R)5′-CTTCTCAGATCCTCTGACAC-3′ SEQ ID NO:47 D20548(F)5′-TCCAGAATCTGTTCCAGAGCGTGC-3′ SEQ ID NO:48 D20548(R)5′-GCTGTGAAGGTTGCTGTTCCTCAT-3′ SEQ ID NO:49 5′-AATCTGGGCGACAAGAGTGA-3′SEQ ID NO:50 5′-ACATCTCCCCTACCGCTATA-3′ SEQ ID NO:515′-TCAGTCTCTCTTTCTCCTTGCA-3′ SEQ ID NO:52 5′-TAGGAGCCTGTGGTCCTGTT-3′ SEQID NO:53 D853G7(F) 5′-GACCCTGTCAAGGAAAGAAAGAGA-3′ SEQ ID NO:54 D853G7(R)5′-CCATTTCAAATTTGGGACCACACTG-3′ SEQ ID NO:55 THO(F)5′-AAGCTGCCCTAGTCAGCAC-3′ SEQ ID NO:56 THO(R) 5′-GCTTCCGAGTGCAGGTCACA-3′SEQ ID NO:57 D115488(F) 5′-mGGAAGGAAGGAAGGAAAGG-3′ SEQ ID NO:58 Dl15488(R) 5′-CTGATAGCCTGACCTGACTGTG-3′ SEQ ID NO:59 D135802(F)5′-CACAGTGAGACTCTATCTCAAAAA-3′ SEQ ID NO:60 D135802(R)5′-TCAGACTGGCTTAGACTGTGG-3′ SEQ ID NO:61 D175695(F)5′-CTGGGCAACAAGAGCAAAATTC--3′ SEQ ID NO:62 D175695(R)5′-mGTTGTTGTTCATTGACTTCAGTCT3′ SEQ ID NO:63 D175654(F)5′-GACCTAGGCCATGTTCACAGCC-3′ SEQ ID NO:64 D175654(R)5′-GACATCCATTGGCACCACCCCAA3′

[0050] Those of ordinary skill in the art will know of variousamplification methodologies which can also be utilized to increase thecopy number of target nucleic acid. Microsatellite DNA sequence detectedin the method of the invention can be further evaluated, detected,cloned, sequenced, and the like, either in solution or after binding toa solid support, by any method usually applied to the detection of aspecific DNA sequence such as another polymerase chain reaction,oligomer restriction (Saiki et al., Bio/Technology 3: 1008-1012 (1985)),allele-specific oligonucleotide (ASO) probe analysis (Conner et al.,Proc. Natl. Acad. Sci. USA 80: 278 (1983), oligonucleotide ligationassays (OLAs) (Landegren et al., Science 241: 1077 (1988)), and thelike. Molecular techniques for DNA analysis have been reviewed(Landegren et al, Science, 242: 229-237 (1988)). Following DNAamplification, the reaction product may be detected by Southern blotanalysis, without using radioactive probes. In such a process, forexample, a small sample of DNA containing a very low level ofmicrosatellite DNA nucleotide sequence is amplified, and analyzed via aSouthern blotting technique. The use of non-radioactive probes or labelsis facilitated by the high level of the amplified signal. In a preferredembodiment of the invention, one nucleoside triphosphate isradioactively labeled, thereby allowing direct visualization of theamplification product by autoradiography. In another embodiment,amplification primers are fluorescent labeled and run through anelectrophoresis system. Visualization of amplified products is by laserdetection followed by computer assisted graphic display.

[0051] In another embodiment, the invention is a method for detecting acell proliferative disorder in a subject. The method detects geneticinstability in a sample of microsatellite DNA of the mammal as anindication of a cell proliferative disorder. As used herein, the term“genetic instability” refers to genetic recombinations which arecharacteristic of tumor cells and which result in nucleic acidmutations. Such mutations include the deletion and addition ofnucleotides. The genetically unstable sequences of the invention arepreferably microsatellite DNA sequences which, by definition, are smalltandem repeat DNA sequences.

[0052] Genetic recombination tends to occur most frequently at regionsof the chromosome where the DNA is homologous (where the DNA has a highdegree of sequence similarity). Where a DNA sequence is repetitive, theDNA homology is greater. The DNA homology occurs not only at the samegenetic locus on the other pair of chromosomes, but also on othergenetic loci or within the same locus on the same chromosome. In normal(non-tumor cells) this genetic recombination tends to be suppressed.Tumor cells, however, characteristically undergo increased geneticrecombination. Where a DNA sequence is repetitive, genetic recombinationcan result in the loss of repeat DNA sequences or the gain of repeat DNAsequences at a genetic locus.

[0053] When the microsatellite DNA repeat is larger, it is more likelythat the microsatellite DNA locus will have mutations. A trinucleotiderepeat is more likely to have deletions or additions than a dinucleotiderepeat. A regular repeat, such as AATAATAAT is more likely to havemutations than a sequence which contains interruptions in the repeatsequence, e.g., AATGACAATAAT. Consequently, those of ordinary skill inthe art can readily identify other target nucleic acid sequences byconsidering the size of the candidate sequence and whether the sequenceis uninterrupted without resorting to undue experimentation. Othermicrosatellite DNA markers will be known by the criteria describedherein and are accessible to those of skill in the art. Smallermicrosatellite DNA markers including dinucleotide and mononucleotiderepeats will also be useful for this analysis.

[0054] The genetic instability may be detected as an amplification ofnucleotide repeats in the DNA. The term “amplification of nucleotiderepeats” refers to a mutation in the sequence of the microsatellite DNAwherein the resulting microsatellite DNA sequence has more DNA repeatsthan the sequence found in normal (non-tumor) cells. Where the normalcell microsatellite DNA has a sequence (X)_(n1), X is the number ofnucleotides, and n1 and n2 are numbers of repeats, the resultingmicrosatellite DNA sequence is (X)_(n1+n2).

[0055] The instability may be detected as a deletion of nucleotiderepeats in the DNA. The term “deletion of nucleotide repeats” refers toa mutation in the sequence of the microsatellite DNA wherein theresulting microsatellite DNA sequence has fewer DNA repeats than thesequence found in normal (non-tumor) cells. Where the normal cellmicrosatellite DNA has a sequence (X)_(n1), X is the number ofnucleotides, n1 and n2 are numbers of repeats, and n1 is greater thann2, the resulting microsatellite DNA sequence is (X)_(n1−n2).

[0056] The instability may be detected when the DNA is amplified beforedetecting. The amplification may be accomplished by the polymerase chainreaction, as described supra. Those of skill in the art will know ofother amplification methods which can increase the copy number of targetnucleic acid.

[0057] The genetic instability may be detected when the cellproliferative disorder is not due to a repair gene defect. The term“repair gene defect” refers to a defect in a gene coding for any of anumber of processes to repair damaged DNA. In gene repair, the damagedportions of the DNA molecule are removed by enzymes (each enzyme codedfor by a repair gene), leaving holes where bases should be. Then otherenzymes (also coded for by repair genes) remove an entire segment ofDNA, in the middle of which was the hole or holes. A DNA polymerase(coded for by repair genes) then fills the gap with nucleotide bases,based on what bases are on the opposite strand of DNA. Finally a ligaseenzyme (coded for by repair genes) seals the phosphate backbone backtogether.

[0058] Another type of DNA repair is error-prone repair (SOS repair),which occurs when both nucleotides in a base pair are missing, such thatit is not possible to maintain accuracy. The enzymes and other proteinswhich mediate error-prone repair are coded for by repair genes. Stillanother type of DNA repair is excision repair, where the damaged portionof DNA is excised, or removed, then the removed part is recopied fromthe other undamaged strand by DNA polymerase enzymes, and finally thereplacement part is attached to the site by DNA ligase enzymes. Theenzymes and other proteins which mediate excision repair are also codedfor by repair genes.

[0059] Those of skill in the art will be familiar with those genes whichcode for the processes which repair damaged DNA. Those of skill in theart will also be able to identify these genes by their chromosomallocation and by the methods of DNA amplification and size fractionation.

[0060] The genetic instability may be detected when the cellproliferative disorder is a neoplasm. Neoplasms are described supra.

[0061] The microsatellite DNA may be from a locus that has been used ingenetic mapping. Among the loci may be one or more of the following:DRPLA, UT762, IFNA, D9S200, D9S156, D3S1284, D3S1238, CHRNB1, D17S86,D9S747, D9S171, D16S476, D4S243, D14S50, D21S1245, FgA, D8S3G7, THO,D115488, D135802, D175695, D175654, and D20548.

[0062] The term “oligonucleotide primer” refers to a sequence of two ormore deoxyribonucleotides or ribonucleotides, preferably at least eight,which sequence is capable of initiating synthesis of a primer extensionproduct that is substantially complementary to a target nucleic acidstrand. The oligonucleotide primer typically contains fifteen totwenty-two or more nucleotides, although it may contain fewernucleotides if the primer is complementary, so as to specifically allowthe amplification of the specifically desired target nucleotidesequence.

[0063] The oligonucleotide primers for use in the invention may beprepared using any suitable method, such as conventional phosphotriesterand phosphodiester methods or automated embodiments thereof. In one suchautomated embodiment, diethylphosphoramidites are used as startingmaterials and may be synthesized as described by Beaucage et al.,Tetrahedron Letter, 22: 1859-1862 (1981). One method for synthesizingoligonucleotides on a modified solid support is described in U.S. Pat.No. 4,458,066. The exact length of primer will depend on many factors,including temperature, buffer, and nucleotide composition. The primermust prime the synthesis of extension products in the presence of theinducing agent for amplification.

[0064] Primers used according to the method of the invention arecomplementary to each strand of mutant nucleotide sequence to beamplified. The term “complementary” means that the primers musthybridize with their respective strands under conditions which allow theagent for polymerization to function. In other words, the primers thatare complementary to the flanking sequences hybridize with the flankingsequences and permit amplification of the nucleotide sequence.Preferably, the 3′ terminus of the primer that is extended has perfectlybase paired complementarity with the complementary flanking strand.

[0065] The term “flanks nucleotide repeats” refers to those DNAsequences on chromosome that are upstream (5′) or downstream (3′) to theDNA sequence to be amplified. The nucleotide repeat sequence to beamplified is preferably a microsatellite DNA sequence. For example, whenthe nucleotide repeat sequence to be amplified is double stranded, afirst sequence that is 5′ to the nucleotide repeat sequence and a secondsequence that is 5′ to the nucleotide repeat sequence on thecomplementary strand flank the microsatellite DNA sequence.

[0066] The nucleotide sequences that flank nucleotide repeats, i.e., thenucleotide sequences to which the oligonucleotide primers hybridize, maybe selected from among the following nucleotide sequences: SEQ IDNO:1-32.

[0067] When it is desirable to amplify the target nucleotide sequence,such as a microsatellite DNA sequence, before detection,oligonucleotides can be used as the primers for amplification. Theoligonucleotide primers are designed based upon identification of thenucleic acid sequence of the flanking regions contiguous with themicrosatellite DNA. One skilled in the art will be able to generateprimers suitable for amplifying target sequences of additional nucleicacids, such as those flanking loci of known microsatellite DNAsequences, using routine skills known in the art and the teachings ofthis invention.

[0068] The oligonucleotide primers used in the amplification may beselected from among the following primers: SEQ ID NO:33-64.

[0069] In another embodiment, the invention provides a kit for detectinga mammalian cell proliferative disorder. The kit comprises anoligonucleotide primer that is complementary to a nucleic acid sequencethat flanks nucleotide repeats of microsatellite DNA. Such a kit mayalso include a carrier means being compartmentalized to receive in closeconfinement one or more containers such as vials, tubes, and the like,each of the containers comprising one of the separate elements to beused in the method. For example, one of the containers may includeamplification primers for a microsatellite DNA locus or a hybridizationprobe, all of which can be detectably labeled. If present, a secondcontainer may comprise a lysis buffer.

[0070] The kit may also have containers containing nucleotides foramplification of the target nucleic acid sequence which may or may notbe labeled, or a container comprising a reporter, such as abiotin-binding protein, such as avidin or streptavidin, bound to areporter molecule, such as an enzymatic, florescent, or radionuclidelabel. The term “detectably labeled deoxyribonucleotide” refers to ameans for identifying deoxyribonucleotide. The detectable label may be aradiolabeled nucleotide. The detectable label may be a small moleculecovalently bound to the nucleotide where the small molecule isrecognized by a well-characterized large molecule. Examples of thesesmall molecules are biotin, which is bound by avidin, and thyroxin,which is bound by anti-thyroxin antibody. Other methods of labeling areknown to those of ordinary skill in the art, including fluorescentcompounds, chemiluminescent compounds, phosphorescent compounds, andbioluminescent compounds.

[0071] In general, the primers used according to the method of theinvention embrace oligonucleotides of sufficient length and appropriatesequence which provide specific initiation of polymerization of asignificant number of nucleic acid molecules containing the targetnucleic acid under the conditions of stringency for the reactionutilizing the primers. In this manner, it is possible to selectivelyamplify the specific target nucleic acid sequence containing the nucleicacid of interest. Oligonucleotide primers used according to theinvention are employed in any amplification process that producesincreased quantities of target nucleic acid.

[0072] The above disclosure generally describes the present invention. Amore complete understanding can be obtained by reference to thefollowing specific examples. These examples are provided herein forpurposes of illustration only and are not intended to limit the scope ofthe invention.

EXAMPLE 1 Molecular Detection of Primary Bladder Cancer byMicrosatellite DNA Analysis

[0073] The purpose of this Example is to show that microsatellite DNAmarkers are useful as clonal markers for the detection of human cancer,because simple DNA repeat mutations can be readily detected in clinicalsamples by the polymerase chain reaction. In this Example, thefeasibility of polymerase chain reaction-based microsatellite DNAanalysis of DNA from urine sediment is shown by correctly identifyingnineteen of twenty patients with primary bladder tumors by thisapproach. In contrast, using urine cytology, only nine of eighteenaffected patients were detected.

[0074] Sixty trinucleotide and tetranucleotide markers in the DNA fromfifty anonymous primary bladder cancers were screened. The screening wasdone in the following manner: Frozen tumor tissue was cut into 10 μmsections. All samples, including lymphocytes, were digested with 1%SDS-proteinase K at 60° C. for 5 hr. DNA was extracted by ethanolprecipitation. Urine samples were spun at 3000 g for 5 min and washedtwice with phosphate-buffered saline. Each polymerase chain reactionmixture (25 μl) contained 50 ng of DNA template. Primers were obtainedfrom Research Genetics (Huntsville, Ala.) or synthesized from sequencesin the Genome Database. For microsatellite DNA analysis, one primer waslabeled with T4 polynucleotide kinase (New England Biolabs) and[γ-³²P]-adenosine triphosphate (New England Nuclear). DNA was subjectedto 30 to 35 cycles of amplification in a Hybaid (Middlesex, UK) OmnigeneTR3 SM2 Thermocycler as follows: 95° C. for 30 sec, 52° to 60° C. for 1min, and 72° C. for 1 min, with a final extension step of 72° C. for 5min. Polymerase chain reaction products were separated byelectrophoresis in denaturing 8 M urea-polyacrylamide-formamide gels,which were then subjected to autoradiography.

[0075] Of the screened primary bladder cancers, 40 (80%) contained atleast one marker alteration when compared with the DNA from matchednormal lymphocytes. Calculations showed that a panel of the ten mostuseful markers would theoretically detect mutations in 52% of allcancers. The calculation was done as follows: The total mutations foreach marker tested in all tumors were tabulated and then grouped, indescending order, from the markers most susceptible in mutations thatwould empirically detect the greater number of primary tumors. Thus, theten most susceptible markers empirically identified at least onalteration in twenty-six of fifty tumors (52%), eleven identified 56% oftumors, twelve identified 60%, thirteen identified 62%, and so forth.Some markers identified mutations only in tumors previously identifiedby another marker, and 20% of tumors did not demonstrate a singlealteration with any marker tested.

[0076] Twenty-five patients who were screened for primary bladdercancers presented with symptoms suggestive of bladder cancer (forexample, gross hematuria) and were found to harbor suspicious lesions atcystoscopy. Urine samples were collected (before cystoscopy) and werethen distributed in blinded fashion for microsatellite DNA analysis androutine urine cytology. Then, the DNA in the urine sediment from thesetwenty-five patients with suspicious bladder lesions and from fivecontrols (patients without evidence of bladder cancer) was tested. Urineand lymphocyte DNA from each patient were amplified by polymerase chainreaction, and polymorphic alleles were compared at the ten preselectedmicrosatellite DNA loci. The urine DNA of ten patients contained amicrosatellite DNA mutations (expansion or deletion of a repeat unit),in close agreement with the frequency expected on the basis of thecalculations.

[0077] In addition to microsatellite DNA mutations, primary tumors oftenharbor chromosomal deletions at suppressor gene loci that are manifestedas loss of heterozygosity loss of heterozygosity and are readilydetected by microsatellite DNA analysis. Notably, eighteen urine DNAsamples also demonstrated loss of heterozygosity, particularly withmarker D9S747 from chromosome 9p21. This result is consistent with theobservation that loss of chromosome 9 occurs frequently in bladdercancer. Analysis of three additional dinucleotide markers on chromosome9p21 (D9S171, D9S162, and IFNA) for loss of heterozygosity confirmed thepresence of deletions in urine samples that demonstrated loss ofchromosome 9 with marker D9S747.

[0078] That the genetically altered alleles were derived from exfoliatedcancer cells was confirmed by two methods:

[0079] First, the primary tumors from biopsies of fifteen of the twentycancer patients (in five cases, there was insufficient biopsy materialfor this analysis) were examined. In all patients, the samemicrosatellite DNA mutations and loss of heterozygosity patternsdetected in the urine were also detected in the primary tumor. However,in two patients, the urine samples showed loss of heterozygosity ormicrosatellite DNA mutations that were not present in the biopsies. Inboth cases, loss of heterozygosity in at least one locus (and loss ofthe identical allele) was shared between the urine sediment and theprimary tumor. Conceivably, the urine sample may have contained a moreadvanced tumor cell clone that was derived from the same progenitor cellbut was not sampled by the small biopsy of the tumor.

[0080] Second, cells from the same urine samples were then examined bylight microscopy. Cytologic analysis was performed in a blinded fashion,following normal clinical procedures in samples from eighteen of thetwenty patients with bladder cancer and from three of the five patientswith suspicious lesions but without neoplasia. Those normal clinicalprocedures are as follows: Approximately 50 cm³ of urine was obtainedand concentrated by centrifugation on cytospin glass slides or Milliporefilters (Burlington, Mass.). Cells were stained by Papanicolaou stainand visualized under microscopy. Standard morphologic criteria were usedto establish the presence of neoplastic cells. Neoplastic cells wereidentified by cytology in nine of the eighteen patients for whommolecular analysis was positive and in one patient for whom molecularanalysis was negative.

[0081] Twenty of the twenty-five patients had histologically confirmedbladder cancer. Overall, microsatellite DNA analysis with the thirteenmarkers detected genetic mutations in nineteen of these twenty cancerpatients. Of four patients with inflammation that prompted cystoscopy,two showed molecular changes (loss of heterozygosity, geneticinstability, or both) in the urine, and both had bladder lesionscontaining atypical cells that were suspicious but not diagnostic forcancer. None of the five patients without neoplasia (controls) showedany microsatellite DNA mutations (see FIGS. 1 and 2).

[0082] This Example demonstrates that microsatellite DNA analysis can bea powerful tool in the detection of primary bladder cancer. The ease ofloss of heterozygosity detection in urine sediment is consistent withanalysis on urine samples by fluorescence in situ hybridization (FISH).Moreover, molecular analysis of patients with multiple tumors hasdemonstrated that these multiple tumors appeared to arise from a singleprogenitor cell that seeded and populated the bladder mucosa,potentially accounting for the high risk of recurrence in thesepatients. These observations are compatible with the hypothesis thatlarge areas of transformed bladder mucosa can exist in patients withsmall neoplasms. Other factors may also contribute to the enrichment oftumor cells in urine; for example, more tumor cells than normal cellsmay survive storage. In addition, as tumor surfaces are composed ofactively growing cell populations that clonally expand throughmechanisms such as loss of adhesion, it is possible that these cells aremore readily shed into the urine.

[0083] These microsatellite DNA markers enabled the detection of 95% ofthe bladder cancers in this study, but as new markers are identified,the approach can be expanded and improved. Despite an expectedidentification of only ˜50% of cases, the identification of loss ofheterozygosity in addition to microsatellite DNA mutations greatlyimproved the detection strategy. An adenocarcinoma of the prostaticfossa was also identified, indicating that markers commonly deleted inother genitourinary tract neoplasms may facilitate the detection ofother neoplasms that exfoliate cells into urine sediment.

[0084] Finally, our approach highlights the immediate utility of studiesthat demonstrate loss of heterozygosity in human cancer and of thedevelopment of molecular progression models for clinical detection. Inmost of the cases in this study, morphologic and cytologic analyses werenot diagnostic. Molecular analysis reliably detected tumors of allgrades and stages, including those often missed by cytology. Inprinciple, this molecular approach can be performed at approximatelyone-third the cost of cytology and does not require exhaustive expertinterpretation. Moreover, the entire assay is amenable tononradioactive, non-gel separation techniques and potentially could leadto a reliable, yet inexpensive, molecular screening test.

EXAMPLE 2 Microsatellite DNA Alterations in Serum DNA of Head and NeckCancer Patients

[0085] The purpose of this Example is to show that microsatellite DNAanalysis of serum represents a novel method for the detection ofcirculating tumor cell DNA. Lymphocyte, serum and tumor DNA wereretrospectively analyzed from twenty-one head and neck cancer patients.Head and neck cancer remains a morbid and often fatal disease. Largetumor bulk and tumor extension are predictors of local regionalrecurrence and poor outcome. Molecular detection of occult neoplasticcells in surrounding surgical margins is a strong predictor of localregional recurrence resulting in a significant decrease in overallsurvival. In this Example, twenty-one patients were followed frominitial diagnosis of transitional cell carcinoma with microsatellite DNAanalysis of urine DNA at the time of cystoscopic evaluations scheduledat routine intervals. In almost all cases, DNA-based analysis correlatedprecisely with clinical findings at cystoscopy and subsequenthistopathology. In two cases, DNA analysis correctly predicted thepresence of a tumor several months before the lesion was detected duringexamination with a cystoscope.

[0086] These twenty-one patients were chosen from our tumor bank becauseall three DNA sources were available for complete analysis, and theserum samples had been collected before surgical resection of head andneck cancer. Twelve microsatellite DNA markers were selected to detectshifts of loss of heterozygosity. Eight markers were chosen on 9p, 3pand 17p, because these chromosomal arms show the highest percentage ofloss of heterozygosity and appear to harbor tumor suppressor gene lociinvolved early in the progression of head and neck cancer. Furthermore,two trinucleotide (D1S50 and DRPLA) and two tetranucleotide (D21S1245and FgA) markers, recognized as being prone to microsatellite DNAinstability and located on loci commonly altered in cancers, were usedin the study for increased sensitivity in the detection of shifts. Lossof heterozygosity was scored if the allele signal was reduced to lessthen 50% of control intensity. Shifts were called if there was anobvious new allele compared with normal (non-tumor) lymphocyte DNA.

[0087] Sample collection and DNA isolation was accomplished as follows:Tumors obtained fresh from surgical resection and blood by venipuncturefrom head and neck cancer patients were collected from patients at theJohns Hopkins University Medical Institutions with prior consent. Toobtain, serum, clotted blood specimens were centrifuged at low speed for5 min, and the serum was stored at −80° C. before DNA extraction. Tumortissue was frozen and microdissected. Lymphocytes, tumor tissue andserum were digested in SDS and proteinase K at 48° C. overnight,followed by phenol/chloroform extraction and ethanol precipitation ofDNA. The mean concentration of DNA in the cancer patients was 110±50 ngper ml serum, and 10 μl was usually sufficient for robust microsatelliteDNA analysis. The concentration of serum DNA from normal controls rangesfrom 0 to 100 ng/ml.

[0088] Polymerase chain reaction amplification was performed as follows:Oligonucleotide markers for microsatellite DNA analysis were obtainedfrom Research Genetics (Huntsville, Ala.) and included IFNA, D9S156,D9S161, D9S200 on 9p; D3S1238, D3S1284 on 3p; D17S786 and CHRNB1 on 17p.Trinucleotide and tetranucleotide primers used included the following:for D14S50: D14S50(F) 5′-AACACCCCTAATTCACCACT-3′ (SEQ ID NO:43)D14550(R) 5′-ATGATTCCACAAGATGGCAG-3′ (SEQ ID NO:44) for D2151245:D21S1245(F) 5′-GTCAGTATTACCCTGTTACCA-3′ (SEQ ID NO:35) D2 151245(R)5′-GTTGAGGATTTTTGCATCAGT-3′ (SEQ ID NO:36) for DRPLA: DRPLA(F)5′-CACAGTCTCAACACATC-3′ (SEQ ID NO:39) DRPLA(R)5′-CCTCCAGTGGGTGGGGAAATGCCTC-3′ (SEQ ID NO:40) for FgA: FgA(F)5′-CCATAGGTTTTGAACTCACAG-3′ (SEQ ID NO:45) FgA(R)5′-CTTCTCAGATTCCTTCTTGACAC-3 (SEQ ID NO:46)

[0089] One primer from each set was end labeled with (γ-³²P) ATP(Amersham) using T4 polynucleotide kinase (New England Biolabs).Polymerase chain reaction amplification was performed with 30-60 ng DNAwas described previously. Products were separated in 8% denaturingurea-polyacrylamide-formamide gels followed by autoradiography. Loss ofheterozygosity was called if the ratio of one allele was significantlydecreased (>50%) in tumor or serum DNA compared with normal (non-tumor)lymphocyte DNA.

[0090] A tumor-specific microsatellite DNA shift, represented by a novelallele after gel electrophoresis, can still be seen when tumor DNA isdiluted between 1:500 to 1:1000 with normal DNA. Shifts derived fromprimary tumor cell DNA in the serum might have been expected. However,there was a surprisingly clear loss of heterozygosity in serum DNA. Thefirst patient that had such provocative results was an 80-year-old mandiagnosed with T₃N_(c)M₀ glottic cancer who underwent a totallaryngectomy in January 1993. He had no evidence of disease on threemonths' follow-up. However, in September 1993, he was diagnosed withrecurrence of tumor in the right neck with the mass surrounding theright carotid artery, and he died of regional disease in November 1994.The serum DNA of this patient displayed a clear loss of heterozygosity.This result was unexpected and reminiscent of the clear loss ofheterozygosity and shifts seen in the urine of patients diagnosed withbladder cancer.

[0091] After microsatellite DNA analysis of all specimens was completed,clinical data were correlated with the results. Clinical correlation wasperformed as follows: Clinical outcome data was obtained from the JohnsHopkins Head and Neck Cancer Tumor Registry and by chart review.Fisher's exact test was used to compare results from plasma analysiswith clinical outcome parameters (see FIGS. 3 and 4).

[0092] All six patients that had microsatellite DNA mutations in theserum DNA demonstrated identical mutations in the primary tumor DNA.Four out of six patients displayed mutations in more than one locus, andall of these patients had advanced disease (stage III-IV). In this smallgroup, four patients went on to die from cancer, one patient hasterminal cancer with metastases and one patient has no evidence ofdisease at three years' follow-up. Five patients had nodal metastasesand three of them later developed distant metastases, one patient tolung and bone and the other two patients to lung and liver.

[0093] Conversely, another nine patients had advanced stage cancer, butno microsatellite DNA mutations in their serum DNA. Six of thesepatients had successful resections and were free of disease on long-termfollow-up (more than one year); two of them died within two yearsdiagnosis from regional recurrence and one was lost to follow-up. Sevenof these patients, including those with a good prognosis, exhibited lossof heterozygosity or shifts in their primary tumor DNA but had noevidence of mutations in serum DNA. Lack of positive findings in serumDNA was also seen in six out of twenty-one patients that had stage I andII cancer, all with good prognoses except for one patient who had arecurrence seven years later and died. Three patients displayed nomicrosatellite DNA mutations in their primary tumor DNA with the testedmarkers. The data are statistically significant for a positive serumtest as predictor of future distant metastases by the Fisher's exacttest (P=0.015); nevertheless, any conclusions of predictive ability orclinical utility require verification with larger populations andwell-defined cohorts.

[0094] The results of this Example support the idea of tumor DNAenrichment in blood serum and plasma. Identification of clear loss ofheterozygosity strongly favors the hypothesis that tumor DNA is enrichedand, in fact, the predominant form of DNA in the plasma. Such analysisof plasma DNA will be useful in follow-up of cancer patients receivingmedical or surgical treatment. Moreover, serum microsatellite DNAmutations were always identical to mutations in the primary tumor DNA.The higher frequency of plasma mutations in small cell lung cancer mayreflect the much higher frequency of clinical metastases in SCLCpatients compared with head and neck cancer patients. In bulky head andneck tumors, cell lysis by necrosis or even apoptosis leads to therelease of naked DNA into the circulation. For large tumors, thisphenomenon may occur more frequently because of local angiogenesis andnecrosis. Although a surprising finding, tumor DNA readily survives invarious bodily fluids including urine, stool and sputum.

EXAMPLE 3 Detection of Bladder Cancer Reoccurrence by Microsatellite DNAAnalysis of Urine

[0095] The purpose of this Example was to demonstrate that themicrosatellite DNA analysis method can be used for following-up patientswith transitional cell carcinoma. A reliable, non-invasive method formonitoring patients with transitional cell carcinoma of the bladderwould be of great clinical benefit. In this Example, serial urinesamples were tested from twenty-one patients who had been treated forbladder cancer with twenty polymorphic microsatellite DNA markers in ablinded fashion. Recurrent lesions were detected in ten out of elevenpatients and correctly predicted the existence of a neoplastic cellpopulation in the urine of two patients, four and six months beforecystoscopic evidence of the tumor. The assay was negative in ten of tenpatients who had no evident cancer. This Example shows thatmicrosatellite DNA analysis of urine sediment represents a novel andpotentially powerful clinical tool for the detection of recurrentbladder cancer.

[0096] The microsatellite DNA analysis was done as follows: DNA wastested from the urine of twenty-one patients with a panel of twentymicrosatellite DNA markers immediately after the initial diagnosis of aprimary bladder tumor. This panel of markers comprised thirteen markersused in our initial study and seven additional selected markers. Elevenof these patients were tested in Example 1 and were included here formonitoring of tumor recurrence.

[0097] Initial urine samples (from before resection) were available fromtwenty of these twenty-one patients with transitional cell carcinoma.Tissue and urine specimens were performed as follows: Twenty patientswith histologically confined bladder carcinoma and one patient withtransitional cell carcinoma of the renal pelvis were enrolled into thestudy. Mean age at time of diagnosis was 68.2 years (50-86), themale/female ratio was 2:1, and the patients were followed for a mean ofeight months (3-18) at our institution. Venous blood (10 ml) wasobtained from every patient for extraction of normal (germline) DNA tobe used as a control. Urine (50 ml) was collected from each patientbefore surgical intervention (transurethral resection or biopsy) andspecimens from the initial surgery or biopsy were frozen at −70° C.immediately before each follow-up cystoscopy, another 50 ml of urine wasobtained from every patient for analysis. The initial urine sample atfirst diagnosis was not available from one patient. All tumors werediagnosed according to the criteria of American Joint Committee onCancer. Urine for cytology was prepared as described previously andcells were stained with standard Papanicolaou stain. The final diagnosisfrom cytopathology (at the Johns Hopkins Hospital) was entered in thestudy.

[0098] DNA extraction was performed as follows: Erythrocytes were lysedby subjecting the blood to TM-solution (5 mM MgCl, 20 mM Tris buffer),and samples were spun at 3000 r.p.m. for 10 min in order to obtain aleukocyte pellet. Urine was also spun at 3000 r.p.m. and the pellet waswashed with phosphate-buffered saline. Tumor specimens were cut into7-μm sections and standard hematoxylin and eosin staining was performed.After confirmation of the diagnosis, neoplastic tissue wasmicrodissected. This material, as well as the leukocytes and the urinecell pellet, were digested with 1% SDS and 50 μg/ml proteinase K for 12hr at 48° C. DNA was obtained from the samples by phenol-chloroformextraction and ethanol precipitation.

[0099] Microsatellite DNA analysis was performed as follows: DNA derivedfrom leukocytes, urine and tumor was analyzed using a panel of twentymicrosatellite DNA markers on different chromosomes (Research Genetics,Huntsville, Ala., and Oncor, Gaithersburg, Md.). This panel containedthe thirteen markers used in our earlier study and seven new markersthat revealed a high rate of loss of heterozygosity and shifts inprimary bladder tumors.

[0100] Chromosomal location, sequences, and annealing temperatures(54-60° C.) of the seven new primer pairs are as follows: for D8S3G7(Chromosomal arm 8p): D8S3G7(F) 5′-GACCCTGTCAAGGAAAGAAAGAGA-3′ (SEQ IDNO: 53) D8S3G7(R) 5′-CCATTTCAAATTTGGGACCACACT G-3′ (SEQ ID NO: 54) forTHO (Chromosomal arm 11q): THO(F) 5′-AAGCTGCCCTAGTCAGCAC-3′ (SEQ ID NO:55) THO(R) 5′-GCTTCCGAGTGCAGGTCACA-3′ (SEQ ID NO: 56) for D115488(Chromosomal arm 11p): D115488(F) 5′-mGGAAGGAAGGAAGGAAAGG-3′ (SEQ ID NO:57) D115488(R) 5′-CTGATAGCCTGACCTGACTGTG-3′ (SEQ ID NO: 58) for D135802(Chromosomal arm 13q): D135802(F) 5′-CACAGTGAGACTCTATCTCAAAAA-3′ (SEQ IDNO: 59) D135802(R) 5′-TCAGACTGGCTTAGACTGTGG-3′ (SEQ ID NO: 60) forD175695 (Chromosomal arm 17p): D175695(F) 5′-CTGGGCAACAAGAGCAAAATTC-3′(SEQ ID NO: 61) D175695(R) 5′-m GTTGTTGTTCATTGACTTCAGTCT-3′ (SEQ ID NO:62) for D175654 (Chromosomal arm 17p): D175654(F)5′-GACCTAGGCCATGTTCACAGCC-3′ (SEQ ID NO: 63) D175654(R)5′-GACATCCATTGGCACCACCCCAA-3′ (SEQ ID NO: 64) for D20548 (Chromosomalarm 20q): D20548(F) 5′-TCCAGTCCCATCTGGATTG-3′ D20548(R)5′-GAAATAAGTGATGCTGTGATG-3′

[0101] The new markers were chosen by empirically screening 50 bladdertumors with 85 tri- and tetra-nucleotide microsatellite DNA markersRates of loss of heterozygosity and shifts (new alleles) were assessedand the best markers were included into this study. Annealingtemperatures, heterozygosity frequency, and length of polymerase chainreaction products from each loci were obtained from the Genome DataBase.

[0102] One primer of each marker pair was end-labeled with [γ-³²P] ATP(Amersham, Arlington Heights, Ill.) using T4-polynucleotide kinase(Gibco BRL, Gaithersburg, Md.). Genomic DNA (50 ng) was subjected to 35polymerase chain reaction cycles at a denaturing temperature of 95° C.for 30 sec, followed by varying annealing temperatures ranging from54-58° C. for 1 min, an extension step at 70° C. for 1 min and a finalextension step at 70° C. for 5 min on Hybaid thermocyclers (Hybaid,Teddington, UK). Polymerase chain reaction products were then separatedin denaturing 7% polyacrylamide-urea-formamide gels. The runningdistances were calculated according to the expected lengths of thepolymerase chain reaction products. Autoradiography was performedovernight at −80° C. with Kodak X-OAAAT scientific imaging film (EastmanKodak, Rochester, N.Y.). Loss of heterozygosity was scored ininformative cases if a significant reduction (>30%) in the ratio of thesignals from the urine and/or tumor alleles was observed in comparisonwith the corresponding normal (germline) alleles in the adjacent lane.

[0103] The results of this Example show the feasibility of the presentinvention. Loss of heterozygosity or mutations by microsatellite DNAanalysis (a positive test) was found in the urine of eighteen (90%) ofthese patients. In each case, the genetic change in urine DNA wasidentical to that identified in the primary tumor. One of the twoaffected patients missed by microsatellite DNA analysis displayed nomutations at any of the twenty tested loci in the initial urine andtumor, but showed loss of heterozygosity at two loci (D16S476 andD9S162) in the follow-up urine sample collected five months precedingthe eventual detection and resection of a recurrent tumor by cystoscopy.The other patient also showed no mutations in his initial urine or tumorsample with this set of markers. Although both of these patients hadsuperficial, low grade tumors, similar tumors from other patientsdemonstrated multiple genetic changes on several chromosomal arms.

[0104] These patients were tested at routine cystoscopic evaluation atapproximately four to six month intervals for up to twenty-six months.Recurrent tumors were observed in eleven of the twenty-one patients. Tenof these eleven patients (91%) were diagnosed correctly bymicrosatellite DNA analysis of the urine DNA collected at the time offollow-up visits. The urine samples of affected patients displayedmultiple genetic changes on different chromosomal arms, confirming a“positive” result at several markers in many cases. These geneticchanges were also identified in the DNA of paired tumor samples fromeach case when available. In two of these patients, a positive testpreceded overt clinical diagnosis by cystoscopy.

[0105] The single false-negative result was from a patient who had asmall pTa,GI recurrent tumor. Although urine samples and tumor biopsiesexhibited multiple DNA changes at the initial presentation, thefollow-up urine (and tumor) samples at five months appeared to be freeof mutations at the screened loci.

[0106] Recurrence-free status was correctly identified at follow-up bymicrosatellite DNA analysis in ten of the ten patients (100%) who weredisease-free after cystoscopy. In nine of these ten cases, geneticmutations present in urine at initial presentation reverted to normal onfollow-up analysis. One elderly patient underwent a bladder-sparingtreatment protocol for a T3 tumor. During multimodality treatment(chemotherapy and radiation therapy), the molecular test remainedpositive in the urine. At six month follow-up, the patient demonstratedno evidence of recurrent disease, and the molecular urine test revertedback to normal.

[0107] Cytologic analysis at the time of the last follow-up, alsoperformed in a blinded fashion, was available from seventeen patients.Neoplastic cells were identified in only one of eight patients (13%)with recurrent lesions (two of the patients with recurrent disease werenot evaluated by cytology). All nine patients without recurrent diseasewere correctly identified as negative by cytology (cytology was notperformed in the two remaining cases).

[0108] In this Example, ten of eleven patients with recurrenttransitional cell carcinoma of the urinary tract were identified bymicrosatellite DNA analysis of the urine collected at the time ofroutine follow-up. Even small tumors of low stage and grade exhibitedmultiple genetic mutations, allowing precise and definitive diagnosis.Most tumors were detected by loss of heterozygosity; however, onepatient was detected by identification of “shifts” (new alleles) alone.The results of this Example support the theory that malignant cells canundergo enrichment by storage; tumor cells may be resistant to apoptosisand may survive longer than normal cells. Moreover, because the tumorsurfaces contain rapidly growing cell populations, with mutations incell-cell adhesion, malignant tumor cells are shed more readily and ingreater numbers into the urine than normal cells.

[0109] The only patient (and the two patients at initial presentation)whose recurrent tumor could not be diagnosed from the urine had a small,low-grade lesion, without any microsatellite DNA abnormalities at thetwenty tested markers. Thus, the molecular test did not miss anypatients with genetic changes present at these markers in the primarytumors. This suggests that the lack of detection of loss ofheterozygosity or shifts due to normal (non-tumor) cell contaminationhas not been a cause of false-negative results. Small Ta lesions showmicrosatellite DNA mutations as often as more invasive lesions, but theyoften harbor fewer regions of loss of heterozygosity. Theoretically, theuse of a higher number of markers might further increase the sensitivityof the test.

[0110] Cytology detected one of eight patients with recurrent diseaseand missed six of seven patients with pTa lesions. Cytology usuallydetects approximately 50% of superficial lesions and in positive casescan help establish the diagnosis before cystoscopy. Failure to detectthese small tumors may probably be less significant clinically.Progression to muscle-invasive tumors occurs in only 3% in this stage,and metastatic spread has been observed in 5% of patients. However,patients with these small lesions may also benefit from chemopreventiveapproaches to prevent progression, in addition to definitive resection.In this regard, molecular analysis appears more promising as a methodfor detecting these early lesions for additional interventions.

[0111] Ten of the ten patients without evidence of recurrent diseasewere diagnosed correctly by microsatellite DNA analysis of the urinesamples. Moreover, all examinations during clinically confirmeddisease-free intervals also served as negative controls for eachpatient. Most patients with bladder cancer will develop recurrencewithin two years, and thus longer follow-up will be necessary in somepatients to confirm the accuracy of molecular diagnosis.

[0112] Two patients were positive several months before clinicalconfirmation by cystoscopy. In these cases, the molecular changesobviously preceded the clinically overt macroscopic findings seen bycytoscopy. In another retrospective study in lung cancer, a positivemolecular test in the sputum of one patient was found thirteen monthsbefore the development of lung cancer.

[0113] The status of recurrent disease was diagnosed correctly in apatient, from the follow-up urine sample with marker D17S695. It wasintriguing that the recurrent tumor had three other distinct molecularmutations, yet none of these could be detected in the urine. Thispatient had multifocal disease. It is probable that the biopsy samplecontained a clonal neoplastic population different from that identifiedin the urine. Biopsies from the other lesions were not available todefinitely ascertain which tumor shed the predominant cell populationinto the urine.

[0114] The data in this Example provide an insight into the potentialusefulness of microsatellite DNA urine analysis for monitoring patientsfor recurrent disease. This Example shows a high sensitivity for thisassay in detecting recurrent tumors, demonstrating the first potentialclinical utility for this approach. Moreover, the use of additionalmarkers capable of identifying other areas of loss of heterozygosity mayfurther improve the sensitivity of this test for bladder cancer. Inaddition to providing a positive or negative test, this assay mayprovide abundant molecular information regarding tumor progression andprognosis (see FIG. 5).

EXAMPLE 4 Analysis of Saliva in Cases of Head and Neck Cancer

[0115] One hundred and five microsatellite DNA markers were screened inprimary lung, and head and neck cancer, to find those most amenable tomicrosatellite DNA shifts. The eight best markers show the highestfrequency of these shifts. These were tested in twenty-one pairedsamples of tumor and saliva in patients with head and neck cancer. Thesaliva samples were obtained by both swabbing and rinsing the mouth ofthe affected patients. In summary, fifteen out of the twenty-one cancercases with just these eight markers were detected. In eight of thesecases, a new allele or shift was identified in both a tumor and saliva,three patients in which there was both loss of heterozygosity andinstability in the saliva and tumor, and four additional patients whichshowed only loss of heterozygosity in both the tumor and in the salivawith these markers. In addition, twenty-two control samples were testedof patients without cancer and found none of these mutations in saliva.

[0116] The invention now being fully described, it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made without departing from the spirit or scope of the invention.

1 64 18 base pairs nucleic acid single linear Genomic DNA 1 CTTGTGTCCCGGCGTCTG 18 19 base pairs nucleic acid single linear Genomic DNA 2CAGCCCAGCA GGACCAGTA 19 21 base pairs nucleic acid single linear GenomicDNA 3 TGGTAACAGT GGAATACTGA C 21 21 base pairs nucleic acid singlelinear Genomic DNA 4 ACTGATGCAA AAATCCTCAA C 21 26 base pairs nucleicacid single linear Genomic DNA 5 GATGGGCAAA CTGCAGGCCT GGGAAG 26 27 basepairs nucleic acid single linear Genomic DNA 6 GCTACAAGGA CCCTTCGAGCCCCGTTC 27 24 base pairs nucleic acid single linear Genomic DNA 7GATGGTGATG TGTTGAGACT GGTG 24 24 base pairs nucleic acid single linearGenomic DNA 8 GAGCATTTCC CCACCCACTG GAGG 24 20 base pairs nucleic acidsingle linear Genomic DNA 9 GTTCTGGATC ACTTCGCGGA 20 20 base pairsnucleic acid single linear Genomic DNA 10 TGAGGATGGT TCTCCCCAAG 20 20base pairs nucleic acid single linear Genomic DNA 11 AGTGGTGAATTAGGGGTGTT 20 20 base pairs nucleic acid single linear Genomic DNA 12CTGCCATCTT GTGGAATCAT 20 21 base pairs nucleic acid single linearGenomic DNA 13 CTGTGAGTTC AAAACCTATG G 21 20 base pairs nucleic acidsingle linear Genomic DNA 14 GTGTCAGAGG ATCTGAGAAG 20 24 base pairsnucleic acid single linear Genomic DNA 15 GCACGCTCTG GAACAGATTC TGGA 2424 base pairs nucleic acid single linear Genomic DNA 16 ATGAGGAACAGCAACCTTCA CAGC 24 20 base pairs nucleic acid single linear Genomic DNA17 TCACTCTTGT CGCCCAGATT 20 20 base pairs nucleic acid single linearGenomic DNA 18 TATAGCGGTA GGGGAGATGT 20 22 base pairs nucleic acidsingle linear Genomic DNA 19 TGCAAGGAGA AAGAGAGACT GA 22 20 base pairsnucleic acid single linear Genomic DNA 20 AACAGGACCA CAGGCTCCTA 20 24base pairs nucleic acid single linear Genomic DNA 21 TCTCTTTCTTTCCTTGACAG GGTC 24 25 base pairs nucleic acid single linear Genomic DNA22 CAGTGTGGTC CCAAATTTGA AATGG 25 19 base pairs nucleic acid singlelinear Genomic DNA 23 GTGCTGACTA GGGCAGCTT 19 20 base pairs nucleic acidsingle linear Genomic DNA 24 TGTGACCTGC ACTCGGAAGC 20 19 base pairsnucleic acid single linear Genomic DNA 25 CCTTTCCTTC CTTCCTTCC 19 22base pairs nucleic acid single linear Genomic DNA 26 CACAGTCAGGTCAGGCTATC AG 22 24 base pairs nucleic acid single linear Genomic DNA 27TTTTTGAGAT AGAGTCTCAC TGTG 24 21 base pairs nucleic acid single linearGenomic DNA 28 CCACAGTCTA AGCCAGTCTG A 21 22 base pairs nucleic acidsingle linear Genomic DNA 29 GAATTTTGCT CTTGTTGCCC AG 22 24 base pairsnucleic acid single linear Genomic DNA 30 AGACTGAAGT CAATGAACAA CAAC 2422 base pairs nucleic acid single linear Genomic DNA 31 GGCTGTGAACATGGCCTAGG TC 22 23 base pairs nucleic acid single linear Genomic DNA 32TTGGGGTGGT GCCAATGGAT GTC 23 18 base pairs nucleic acid single linearoligonucleotide 33 CAGACGCCGG GACACAAG 18 19 base pairs nucleic acidsingle linear oligonucleotide 34 TACTGGTCCT GCTGGGCTG 19 21 base pairsnucleic acid single linear oligonucleotide 35 GTCAGTATTA CCCTGTTACC A 2121 base pairs nucleic acid single linear oligonucleotide 36 GTTGAGGATTTTTGCATCAG T 21 26 base pairs nucleic acid single linear oligonucleotide37 CTTCCCAGGC CTGCAGTTTG CCCATC 26 27 base pairs nucleic acid singlelinear oligonucleotide 38 GAACGGGGCT CGAAGGGTCC TTGTAGC 27 24 base pairsnucleic acid single linear oligonucleotide 39 CACCAGTCTC AACACATCAC CATC24 24 base pairs nucleic acid single linear oligonucleotide 40CCTCCAGTGG GTGGGGAAAT GCTC 24 20 base pairs nucleic acid single linearoligonucleotide 41 TCCGCGAAGT GATCCAGAAC 20 20 base pairs nucleic acidsingle linear oligonucleotide 42 CTTGGGGAGA ACCATCCTCA 20 20 base pairsnucleic acid single linear oligonucleotide 43 AACACCCCTA ATTCACCACT 2020 base pairs nucleic acid single linear oligonucleotide 44 ATGATTCCACAAGATGGCAG 20 21 base pairs nucleic acid single linear oligonucleotide45 CCATAGGTTT TGAACTCACA G 21 20 base pairs nucleic acid single linearoligonucleotide 46 CTTCTCAGAT CCTCTGACAC 20 24 base pairs nucleic acidsingle linear oligonucleotide 47 TCCAGAATCT GTTCCAGAGC GTGC 24 24 basepairs nucleic acid single linear oligonucleotide 48 GCTGTGAAGGTTGCTGTTCC TCAT 24 20 base pairs nucleic acid single linearoligonucleotide 49 AATCTGGGCG ACAAGAGTGA 20 20 base pairs nucleic acidsingle linear oligonucleotide 50 ACATCTCCCC TACCGCTATA 20 22 base pairsnucleic acid single linear oligonucleotide 51 TCAGTCTCTC TTTCTCCTTG CA22 20 base pairs nucleic acid single linear oligonucleotide 52TAGGAGCCTG TGGTCCTGTT 20 24 base pairs nucleic acid single linearoligonucleotide 53 GACCCTGTCA AGGAAAGAAA GAGA 24 25 base pairs nucleicacid single linear oligonucleotide 54 CCATTTCAAA TTTGGGACCA CACTG 25 19base pairs nucleic acid single linear oligonucleotide 55 AAGCTGCCCTAGTCAGCAC 19 20 base pairs nucleic acid single linear oligonucleotide 56GCTTCCGAGT GCAGGTCACA 20 19 base pairs nucleic acid single linearoligonucleotide 57 GGAAGGAAGG AAGGAAAGG 19 22 base pairs nucleic acidsingle linear oligonucleotide 58 CTGATAGCCT GACCTGACTG TG 22 24 basepairs nucleic acid single linear oligonucleotide 59 CACAGTGAGACTCTATCTCA AAAA 24 21 base pairs nucleic acid single linearoligonucleotide 60 TCAGACTGGC TTAGACTGTG G 21 22 base pairs nucleic acidsingle linear oligonucleotide 61 CTGGGCAACA AGAGCAAAAT TC 22 24 basepairs nucleic acid single linear oligonucleotide 62 GTTGTTGTTCATTGACTTCA GTCT 24 22 base pairs nucleic acid single linearoligonucleotide 63 GACCTAGGCC ATGTTCACAG CC 22 23 base pairs nucleicacid single linear oligonucleotide 64 GACATCCATT GGCACCACCC CAA 23

I claim:
 1. A method for detecting a cell proliferative disorder in asubject, the method comprising: a) obtaining from the subject a samplecomprising microsatellite DNA; and b) detecting an allelic imbalance,wherein an allelic imbalance is indicative of a cell proliferativedisorder.
 2. The method of claim 1, wherein the allelic imbalance isdetected as a decrease in the level of DNA corresponding to an allele.3. The method of claim 2, wherein the level of DNA corresponding to theallele is less than 50% of the level of DNA of a corresponding allele ina microsatellite DNA sample of a subject that lacks the cellproliferative disorder.
 4. The method of claim 1, wherein the allelicimbalance is detected as an increase in the level of DNA correspondingto an allele.
 5. The method of claim 4, wherein the increase is detectedas the presence of a new allele.
 6. The method of claim 1, whereindetection comprises size fractionation of the DNA.
 7. The method ofclaim 6, wherein the DNA is fractionated by gel electrophoresis.
 8. Themethod of claim 1, wherein the cell proliferative disorder is aneoplasm.
 9. The method of claim 8, wherein the neoplasm is selectedfrom the group consisting of head, neck, lung, esophageal, stomach,small bowel, colon, bladder, kidney, and cervical neoplasms.
 10. Themethod of claim 1, wherein the sample is selected from the groupconsisting of urine, sputum, bile, stool, cervical tissue, saliva,tears, cerebral spinal fluid, serum, plasma, and lymphocytes.
 11. Themethod of claim 1, wherein the DNA is amplified by means of nucleotideswhich hybridize to the flanking regions of the microsatellite DNA beforedetecting.
 12. The method of claim 11, wherein the amplificationcomprises a polymerase chain reaction.
 13. The method of claim 1,wherein the microsatellite DNA comprises a locus selected from the groupconsisting of DRPLA, UT762, IFNA, D9S200, D9S156, D3S1284, D3S1238,CHRNB1, D17S86, D9S747, D9S171, D16S476, D4S243, D14S50, D21S1245, FgA,D8S3G7, THO, D115488, D135802, D175695, D175654, and D20548.
 14. Themethod of claim 11, wherein the nucleotide sequences of the flankingregion to which the oligonucleotide primers hybridize are selected fromthe group consisting of SEQ ID NO:1-31 and SEQ ID NO:32.
 15. The methodof claim 14, wherein oligonucleotide primers are selected from the groupconsisting of SEQ ID NO:33-63 and SEQ ID NO:64.
 16. A method fordetecting a cell proliferative disorder in a subject, the methodcomprising: a) obtaining a sample from the subject of microsatelliteDNA; and b) detecting genetic instability in the sample ofmicrosatellite DNA, wherein genetic instability is indicative of a cellproliferative disorder.
 17. The method of claim 16, wherein theinstability is detected as an amplification of nucleotide repeats in theDNA.
 18. The method of claim 16, wherein the instability is detected asa deletion of nucleotide repeats in the DNA.
 19. The method of claim 16,wherein the DNA is amplified before detecting.
 20. The method of claim16, wherein the cell proliferative disorder is not due to a repair genedefect.
 21. The method of claim 20, wherein the cell proliferativedisorder is a neoplasm.
 22. The method of claim 20, wherein the neoplasmis selected from the group consisting of the head, neck, lung, andbladder neoplasms.
 23. The method of claim 21, wherein the neoplasm isbenign.
 24. The method of claim 21, wherein the neoplasm is malignant.25. The method of claim 16, wherein the sample is selected from thegroup consisting of urine, sputum, bile, stool, cervical tissue, saliva,tears, cerebral spinal fluid, serum, plasma, and lymphocytes.
 26. A kitfor detecting a cell proliferative disorder, the kit comprisingoligonucleotide primers that are complementary to a nucleotide sequencethat flanks nucleotide repeats of microsatellite DNA.
 27. The kit ofclaim 26, further comprising a detectably labeled deoxyribonucleotide.28. The kit of claim 26, wherein nucleotide sequences that flanknucleotide repeats of microsatellite DNA to which the oligonucleotideprimers are complementary are selected from the group consisting of SEQID NO:1-31 and SEQ ID NO:32.
 29. The kit of claim 28, wherein theoligonucleotide primers are selected from the group consisting of SEQ IDNO:33-63 and SEQ ID NO:64.